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icc srsf1  (Proteintech)

 
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    Proteintech icc srsf1
    (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. <t>SRSF1</t> and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.
    Icc Srsf1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/icc srsf1/product/Proteintech
    Average 93 stars, based on 1 article reviews
    icc srsf1 - by Bioz Stars, 2025-03
    93/100 stars

    Images

    1) Product Images from "In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity"

    Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

    Journal: bioRxiv

    doi: 10.1101/2021.01.08.425998

    (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. SRSF1 and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.
    Figure Legend Snippet: (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. SRSF1 and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.

    Techniques Used: Construct, Expressing, Immunoprecipitation, Mass Spectrometry, Protein Enrichment, Activation Assay, Two Tailed Test

    (A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype. (B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/genotype). **p < 0.01; ***p < 0.001; ****p < 0.0001. [NTC = non targeting control] (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas- (CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions. (D) Representative photographs of fly eyes as in (C) and quantitation as in (B) with SRSF1/dSF2 overexpression (dSF2 OE). (E) Survival assay on flies expressing (CGG)90-EGFP under Tub5-GS driver with drug initiation starting 1 day post eclosion and continuing through experiment (Log-rank Mantel–Cox test; n = 78-80/genotype) with SRSF1 knockdown *p < 0.05. (F) Representative photographs of fly eyes as in (C) and quantitation as in (B) with siRNA mediated knockdown of SRPK1 (dSRPK1). Details of fly genotypes described in supplementary Table 2.
    Figure Legend Snippet: (A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype. (B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/genotype). **p < 0.01; ***p < 0.001; ****p < 0.0001. [NTC = non targeting control] (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas- (CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions. (D) Representative photographs of fly eyes as in (C) and quantitation as in (B) with SRSF1/dSF2 overexpression (dSF2 OE). (E) Survival assay on flies expressing (CGG)90-EGFP under Tub5-GS driver with drug initiation starting 1 day post eclosion and continuing through experiment (Log-rank Mantel–Cox test; n = 78-80/genotype) with SRSF1 knockdown *p < 0.05. (F) Representative photographs of fly eyes as in (C) and quantitation as in (B) with siRNA mediated knockdown of SRPK1 (dSRPK1). Details of fly genotypes described in supplementary Table 2.

    Techniques Used: Construct, Quantitation Assay, Expressing, Over Expression, Clonogenic Cell Survival Assay

    (A) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs to fly SRSF proteins at 25°C. (B) Quantitation of GMR-GAL4 driven (GGGGCC)28-EGFP eye phenotype with SRSF modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ****p < 0.0001 (C) Representative photographs of fly eyes expressing either GMR-GAL4 driven (GGGGCC)28-EGFP at 29°C with siRNA against SRSF1. Quantification of eye necrosis (D) and eye diameter (E) at 29°C in GMR-GAL4 driven (GGGGCC)28-EGFP flies. (F) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel–Cox test; n = 71-90/genotype) with control or SRSF1 siRNA. **p < 0.01 (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (H) Quantitation of rough eye phenotypes.. t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ***p < 0.001; ****p < 0.0001
    Figure Legend Snippet: (A) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs to fly SRSF proteins at 25°C. (B) Quantitation of GMR-GAL4 driven (GGGGCC)28-EGFP eye phenotype with SRSF modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ****p < 0.0001 (C) Representative photographs of fly eyes expressing either GMR-GAL4 driven (GGGGCC)28-EGFP at 29°C with siRNA against SRSF1. Quantification of eye necrosis (D) and eye diameter (E) at 29°C in GMR-GAL4 driven (GGGGCC)28-EGFP flies. (F) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel–Cox test; n = 71-90/genotype) with control or SRSF1 siRNA. **p < 0.01 (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (H) Quantitation of rough eye phenotypes.. t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ***p < 0.001; ****p < 0.0001

    Techniques Used: Expressing, Quantitation Assay

    (A) Schematic of +1CGG RAN translation nLuc-3xFLAG and AUG-driven control nLuc-3xFLAG reporters. (B) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=10-14) following knockdown of SRSF1. Comparisons between siEGFP and siSRSF1 treated cells. (C) Anti-FLAG Western blot of FMRpolyG nLUC-3xFLAG with and without SRSF1 knockdown in HEK293T cells (n=3). Error bars represent mean +/−SEM. t-test with Welch corrections. *p < 0.05 (D) Representative images of subcellular distribution of CGG-nLuc-3xFLAG reporter RNAs using HCR with and without SRSF1 knockdown in HEK293T cells. n= # of cells used for quantification for each condition as mentioned in the table. Quantification of CGG-nLuc-3xFLAG reporter RNA intensity (E) and nuclear/cytoplasmic distribution (F). Error bars represent mean +/−SEM. t-test with Bonferroni and Welch’s correction *p < 0.05, ****p < 0.0001.
    Figure Legend Snippet: (A) Schematic of +1CGG RAN translation nLuc-3xFLAG and AUG-driven control nLuc-3xFLAG reporters. (B) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=10-14) following knockdown of SRSF1. Comparisons between siEGFP and siSRSF1 treated cells. (C) Anti-FLAG Western blot of FMRpolyG nLUC-3xFLAG with and without SRSF1 knockdown in HEK293T cells (n=3). Error bars represent mean +/−SEM. t-test with Welch corrections. *p < 0.05 (D) Representative images of subcellular distribution of CGG-nLuc-3xFLAG reporter RNAs using HCR with and without SRSF1 knockdown in HEK293T cells. n= # of cells used for quantification for each condition as mentioned in the table. Quantification of CGG-nLuc-3xFLAG reporter RNA intensity (E) and nuclear/cytoplasmic distribution (F). Error bars represent mean +/−SEM. t-test with Bonferroni and Welch’s correction *p < 0.05, ****p < 0.0001.

    Techniques Used: Expressing, Western Blot

    (A) Schematic of AKT/SRPK1 signaling pathway that regulate SRSF1 localization with example of known pharmacological compounds that inhibit SRPK1. (B) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3). (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9) following treatment with DMSO and SRPIN340. (D) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. (E) Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3). (F) Anti-FLAG Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Error bars represent mean +/− SD. *p < 0.05; **p < 0.01 and ***p < 0.001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in the sample buffer.
    Figure Legend Snippet: (A) Schematic of AKT/SRPK1 signaling pathway that regulate SRSF1 localization with example of known pharmacological compounds that inhibit SRPK1. (B) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3). (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9) following treatment with DMSO and SRPIN340. (D) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. (E) Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3). (F) Anti-FLAG Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Error bars represent mean +/− SD. *p < 0.05; **p < 0.01 and ***p < 0.001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in the sample buffer.

    Techniques Used: Western Blot, Expressing



    Similar Products

    icc srsf1  (Proteintech)
    93
    Proteintech icc srsf1
    (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. <t>SRSF1</t> and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.
    Icc Srsf1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/icc srsf1/product/Proteintech
    Average 93 stars, based on 1 article reviews
    icc srsf1 - by Bioz Stars, 2025-03
    93/100 stars
    		  Buy from Supplier

    Image Search Results


    (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. SRSF1 and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.

    Journal: bioRxiv

    Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

    doi: 10.1101/2021.01.08.425998

    Figure Lengend Snippet: (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. SRSF1 and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.

    Article Snippet: For HCR: - Primary Antibodies for ICC - SRSF1 1:100, rabbit from Proteintech, 12929-2-AP; Secondary antibodies for ICC - 1:500, Alexa Fluor 488 goat anti-rabbit IgG from Thermo Fisher Scientific, A11008.

    Techniques: Construct, Expressing, Immunoprecipitation, Mass Spectrometry, Protein Enrichment, Activation Assay, Two Tailed Test

    (A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype. (B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/genotype). **p < 0.01; ***p < 0.001; ****p < 0.0001. [NTC = non targeting control] (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas- (CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions. (D) Representative photographs of fly eyes as in (C) and quantitation as in (B) with SRSF1/dSF2 overexpression (dSF2 OE). (E) Survival assay on flies expressing (CGG)90-EGFP under Tub5-GS driver with drug initiation starting 1 day post eclosion and continuing through experiment (Log-rank Mantel–Cox test; n = 78-80/genotype) with SRSF1 knockdown *p < 0.05. (F) Representative photographs of fly eyes as in (C) and quantitation as in (B) with siRNA mediated knockdown of SRPK1 (dSRPK1). Details of fly genotypes described in supplementary Table 2.

    Journal: bioRxiv

    Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

    doi: 10.1101/2021.01.08.425998

    Figure Lengend Snippet: (A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype. (B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/genotype). **p < 0.01; ***p < 0.001; ****p < 0.0001. [NTC = non targeting control] (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas- (CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions. (D) Representative photographs of fly eyes as in (C) and quantitation as in (B) with SRSF1/dSF2 overexpression (dSF2 OE). (E) Survival assay on flies expressing (CGG)90-EGFP under Tub5-GS driver with drug initiation starting 1 day post eclosion and continuing through experiment (Log-rank Mantel–Cox test; n = 78-80/genotype) with SRSF1 knockdown *p < 0.05. (F) Representative photographs of fly eyes as in (C) and quantitation as in (B) with siRNA mediated knockdown of SRPK1 (dSRPK1). Details of fly genotypes described in supplementary Table 2.

    Article Snippet: For HCR: - Primary Antibodies for ICC - SRSF1 1:100, rabbit from Proteintech, 12929-2-AP; Secondary antibodies for ICC - 1:500, Alexa Fluor 488 goat anti-rabbit IgG from Thermo Fisher Scientific, A11008.

    Techniques: Construct, Quantitation Assay, Expressing, Over Expression, Clonogenic Cell Survival Assay

    (A) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs to fly SRSF proteins at 25°C. (B) Quantitation of GMR-GAL4 driven (GGGGCC)28-EGFP eye phenotype with SRSF modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ****p < 0.0001 (C) Representative photographs of fly eyes expressing either GMR-GAL4 driven (GGGGCC)28-EGFP at 29°C with siRNA against SRSF1. Quantification of eye necrosis (D) and eye diameter (E) at 29°C in GMR-GAL4 driven (GGGGCC)28-EGFP flies. (F) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel–Cox test; n = 71-90/genotype) with control or SRSF1 siRNA. **p < 0.01 (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (H) Quantitation of rough eye phenotypes.. t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ***p < 0.001; ****p < 0.0001

    Journal: bioRxiv

    Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

    doi: 10.1101/2021.01.08.425998

    Figure Lengend Snippet: (A) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs to fly SRSF proteins at 25°C. (B) Quantitation of GMR-GAL4 driven (GGGGCC)28-EGFP eye phenotype with SRSF modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ****p < 0.0001 (C) Representative photographs of fly eyes expressing either GMR-GAL4 driven (GGGGCC)28-EGFP at 29°C with siRNA against SRSF1. Quantification of eye necrosis (D) and eye diameter (E) at 29°C in GMR-GAL4 driven (GGGGCC)28-EGFP flies. (F) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel–Cox test; n = 71-90/genotype) with control or SRSF1 siRNA. **p < 0.01 (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (H) Quantitation of rough eye phenotypes.. t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ***p < 0.001; ****p < 0.0001

    Article Snippet: For HCR: - Primary Antibodies for ICC - SRSF1 1:100, rabbit from Proteintech, 12929-2-AP; Secondary antibodies for ICC - 1:500, Alexa Fluor 488 goat anti-rabbit IgG from Thermo Fisher Scientific, A11008.

    Techniques: Expressing, Quantitation Assay

    (A) Schematic of +1CGG RAN translation nLuc-3xFLAG and AUG-driven control nLuc-3xFLAG reporters. (B) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=10-14) following knockdown of SRSF1. Comparisons between siEGFP and siSRSF1 treated cells. (C) Anti-FLAG Western blot of FMRpolyG nLUC-3xFLAG with and without SRSF1 knockdown in HEK293T cells (n=3). Error bars represent mean +/−SEM. t-test with Welch corrections. *p < 0.05 (D) Representative images of subcellular distribution of CGG-nLuc-3xFLAG reporter RNAs using HCR with and without SRSF1 knockdown in HEK293T cells. n= # of cells used for quantification for each condition as mentioned in the table. Quantification of CGG-nLuc-3xFLAG reporter RNA intensity (E) and nuclear/cytoplasmic distribution (F). Error bars represent mean +/−SEM. t-test with Bonferroni and Welch’s correction *p < 0.05, ****p < 0.0001.

    Journal: bioRxiv

    Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

    doi: 10.1101/2021.01.08.425998

    Figure Lengend Snippet: (A) Schematic of +1CGG RAN translation nLuc-3xFLAG and AUG-driven control nLuc-3xFLAG reporters. (B) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=10-14) following knockdown of SRSF1. Comparisons between siEGFP and siSRSF1 treated cells. (C) Anti-FLAG Western blot of FMRpolyG nLUC-3xFLAG with and without SRSF1 knockdown in HEK293T cells (n=3). Error bars represent mean +/−SEM. t-test with Welch corrections. *p < 0.05 (D) Representative images of subcellular distribution of CGG-nLuc-3xFLAG reporter RNAs using HCR with and without SRSF1 knockdown in HEK293T cells. n= # of cells used for quantification for each condition as mentioned in the table. Quantification of CGG-nLuc-3xFLAG reporter RNA intensity (E) and nuclear/cytoplasmic distribution (F). Error bars represent mean +/−SEM. t-test with Bonferroni and Welch’s correction *p < 0.05, ****p < 0.0001.

    Article Snippet: For HCR: - Primary Antibodies for ICC - SRSF1 1:100, rabbit from Proteintech, 12929-2-AP; Secondary antibodies for ICC - 1:500, Alexa Fluor 488 goat anti-rabbit IgG from Thermo Fisher Scientific, A11008.

    Techniques: Expressing, Western Blot

    (A) Schematic of AKT/SRPK1 signaling pathway that regulate SRSF1 localization with example of known pharmacological compounds that inhibit SRPK1. (B) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3). (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9) following treatment with DMSO and SRPIN340. (D) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. (E) Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3). (F) Anti-FLAG Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Error bars represent mean +/− SD. *p < 0.05; **p < 0.01 and ***p < 0.001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in the sample buffer.

    Journal: bioRxiv

    Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

    doi: 10.1101/2021.01.08.425998

    Figure Lengend Snippet: (A) Schematic of AKT/SRPK1 signaling pathway that regulate SRSF1 localization with example of known pharmacological compounds that inhibit SRPK1. (B) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3). (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9) following treatment with DMSO and SRPIN340. (D) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. (E) Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3). (F) Anti-FLAG Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Error bars represent mean +/− SD. *p < 0.05; **p < 0.01 and ***p < 0.001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in the sample buffer.

    Article Snippet: For HCR: - Primary Antibodies for ICC - SRSF1 1:100, rabbit from Proteintech, 12929-2-AP; Secondary antibodies for ICC - 1:500, Alexa Fluor 488 goat anti-rabbit IgG from Thermo Fisher Scientific, A11008.

    Techniques: Western Blot, Expressing